The calcium-activated potassium current is thought to play a major role in the regulation of cell membrane excitability and pacemaker activity in a wide variety of cell types. The K channels which underlie this current will be studied using patch clamp techniques on excised patches of membrane from cultured rat skeletal muscle to resolve the current flow through individual ion channels. The specific aims are directed towards elucidating the underlying molecular mechanism by testing the Ca-activated K channel's response to step changes in membrane potential, and by characterizing the steady-state channel activity when the membrane potential is held constant. The voltage steps will be delivered by an on-line computer, and the single channel currents will be digitized for later analysis. By averaging the single channel currents elicited by voltage steps, the on- and off-relaxations can be studied and used to test specific kinetic mechanisms of the channel gating. The steady-state activity of the channel will be described in terms of the distributions of open and shut intervals; these distributions are intimately related to the underlying molecular mechanism. The interaction between Ca and the voltage responses will also be studied. In addition, I will follow up preliminary experiments suggesting that the amino acids alanine, glutamate and lysine increase channel activity when Ca is present. These experiments will provide significant information about the molecular mechanism which controls the gating of the Ca-activated K channel, extending and complementing my previous studies of the Ca sensitivity of the channel. Since the channel functions in the intact cell in response to changing Ca and voltage, the experiments described here are consistent with the long-term objectives of understanding how the channel is suited to performing its physiologically important role in the control of cell excitability. In addition, intracellular regulation of both the Ca and amino acid concentrations might provide a direct link between membrane excitability and the metabolic state of the cell which can be studied at the molecular level.